Fluorescence applications, which penetrate nearly every field of biological research, rely on high-quantum yield fluorescent probes such as small-molecular weight organic compounds. Despite their demonstrated utility in advancing our understanding of biological mechanism and serving as important diagnostic tools, the overall utility of such fluorophores is often limited by their stability in biological environments. In particular, the performance of each small-molecule fluorophore class has been shown to be significantly hampered by undesirable photophysical properties that limit both the flux of photons generated as well as the total time interval over which photon emission events can be observed. Such phenomena, which include both transient (blinking) and irreversible (photobleaching), add uncertainties to all fluorescence applications, and are particularly limiting for single-molecule fluorescence studies, where relatively high levels of illumination intensity must be employed. Previously, we have described the characterization of solution additives, which have now come into increasingly widespread use, that provide a means of mitigating the blinking and photobleaching propensities of organic fluorophores. The inclusion of such compounds in biological imaging experiments has provided an effective strategy for enhancing the time resolution and signal-to-noise ratio of single-molecule imaging in both in vitro and in vivo settings by reducing dark state lifetimes and the rate of photobleaching. However, several key limitations hamper their overall utility: 1] they exhibit limited aqueous solubility; 2] they disply poor membrane permeability; and 3] they have potentially toxic side effects that must be carefully considered. Moreover, the benefits of adding protective agents must be empirically determined for each system investigated and their mechanisms of action are not fully understood. Both considerations hamper further advancements. Here, we aim to build on this nascent technology to develop the synthesis of novel fluorescent probes to achieve greater control over their photophysical properties. The anticipated outcome of this research is a suite of novel imaging tools that exhibit enhanced overall performance to enable new areas of investigation over a broad range of in vitro and in vivo applications. The proposed research will also lead to a deeper understanding of the parameters presently limiting fluorophore performance. Novel organic fluorophore derivatives have already been synthesized and characterized that exhibit up to a 20-fold increase in performance over commercially-available material. Beneficial enhancements, are also observed in fully oxygenated solutions. As exemplified in our recent publication in Nature Methods, such fluorophores enable important biological imaging experiments that would have otherwise been impossible to achieve (Altman et al., Nature Methods 2011). Collaborative efforts aimed at understanding the mechanisms of fluorophore protection is anticipated to generate further advancements and the synthesis of next-generation fluorophores that are required to enable otherwise impossible fluorescence imaging applications both in vitro and within living cells. PUBLIC HEALTH RELEVANCE: The proposed research aims to develop next-generation fluorescent probes for biological research, which exhibit superior brightness and stability, in order to enable the burgeoning field of fluorescence imaging both in vitro and within living cells.